A number of methods can be used to purify antibodies and other biomolecules. Purification is performed in solution. Tangential Flow Filtration (TFF) and Affinity Chromatography are commonly employed techniques which retrieve antibodies or other biomolecules from crude process liquors. Size Exclusion Chromatography and Ion Exchange Chromatography are further purification techniques which concentrate and or purify process liquors containing biomolecules. However, all these techniques are themselves wasteful in terms of generating large volumes of waste.
In Tangential Flow Filtration the biomolecule can be retained on a semi-permeable membrane (retentate) not through a chemical bond but via pressure or intermolecular forces. Small molecular weight contaminants are not retained on the membrane and are removed as a permeate stream (filtrate). These contaminants typically include additives purposefully introduced to the process stream. These additives create a stable environment for the biomolecule during the purification process (e.g. buffer salts, detergents, disaccharide stabilisers, preservatives, etc.). Once the biomolecule has been retained it is washed off the membrane with a buffer. This technique has several limitations, which include:                Poor affinity of the biomolecule for membrane (some product is lost in filtrate it it is not retained)        Biomolecule is subject to trans membrane pressure (TMP) which can cause undesired aggregation        Contamination of process liquor due to leachables from TFF membrane        Loss of biomolecule to membrane through ‘blinding’ (if it binds it cannot be removed and further separation becomes increasing difficult)        Fluctuations in retained biomolecule concentration on membrane can lead to the biomolecule exceeding its solubility limit (which leads to precipitation and loss to the membrane)        Effectiveness of process is compromised by minute changes in pH and ionic strength        Loss of product through dead volume and pipework (because the biomolecule is not removed in a concentrated slug of solvent)        Poor selectivity        No differentiation between active biomolecule and fragments or aggregates of the biomolecule        Requires specialist equipment with high cost association that is complicated to set up        
Alternatively, a biomolecule may be purified by affinity purification. Affinity chromatography is based on attaching a ligand to chromatography media, such as an agarose support. For example, to purify antibodies using affinity chromatography a ligand such as Protein A or Protein G can be employed. These ligands have a high affinity for antibodies but do not form a chemical bond to the antibody. Instead they form weak, temporary interactions which are hypersensitive to changes in pH, temperature, media and physical agitation.
Affinity chromatography is often seen as a last resort within pharmaceutical and biomolecule manufacturing and purification due to the high costs associated with operations. These costs accrue due to the cost of preparing the Protein A and Protein G ligands and attaching them to the chromatography media in such a way that affinity is still inferred. As such affinity media are expensive.
Furthermore, the loading of the chromatography media (essentially how much biomolecule, e.g. an antibody, may be attached to the support per gram) is very low; typically micromoles (μM) per gram. To manufacture Kgs of antibody at commercial scale, the cost of the chromatography media required would be prohibitive. Another disadvantage is that Protein A and Protein G affinity media are highly cross-linked Agarose supports which are easily broken by stirring or any form of agitation. Particulates arising from broken beads are a source of contamination and must also be removed from the process liquor during purification.
Many biomolecules are stored at low temperatures such as between 1° C. to 5° C. for short periods or frozen at temperatures below −20° C. for prolonged periods. Many biomolecules are prone to degradation by changes in temperature (the so-called freeze-thaw issue). For example, storing an antibody in a concentrated form in solution, where the antibody has the ability to interact and adopt different conformational positions, can lead to it losing activity. Storing in solution may also increase the risk of environmental exposure to microbial contamination and or endotoxins which is known as a detrimental issue.
An alternative technique for processing biomolecules is solid phase synthesis. This technique can be used in the manufacture of peptides, oligonucleotides and oligosaccharides.
Antibodies—typically used as therapeutic or diagnostic agents—are difficult to make and purify, with low-yielding manufacturing steps & wasteful processes.
At present, antibodies are manufactured by fermentation processes using batch bioreactors. A typical concentration of an antibody process stream is around 0.1% w/v. Therefore, within antibody production there are huge volumes of process liquors that require purification and eventual disposal of the waste generated for such processing. As such, 1 kg of antibody may need a large facility with 1000 liter reactors and associated ancillary equipment. For high potency antibody-based drugs such as Antibody Drug Conjugates (ADCs) the plant needs to be operated within a very high level of airborne containment (typically <50 nano gramme/m3 air measured over a standard 8 hr work day) to prevent exposure of plant operators. Most existing ADC facilities are only able to operate at 50-100 liters scale, limiting the production batch size to 100 s of grammes at very high cost per gramme. For supply of launched products, which may require 10 s to 100 s of kilograms of drug, the problem is multiplied at least ten-fold. Furthermore in using this technique it is not unknown for an expensive batch of antibody to be lost through, for example, an operator error or unforeseen failure of a piece of equipment.
It is therefore an aim of the present invention to provide a system that would reduce the amount of biomolecules lost during processing, purification and/or storage of biomolecules. Ideally such a system would make it physically difficult to lose biomolecules during processing, purification and/or storage. Ideally the system would be highly selective for the biomolecule.
It is also an aim of the present invention to provide a system for processing, purifying and/or storing biomolecules that would not alter the integrity or biological activity of the biomolecule. Ideally, the system would not modify the biomolecule or alter its 3D structure. It is also an aim of the present invention to provide a system for processing, purifying and/or storing biomolecules that would reduce the cost of processing, purification and/or storage of biomolecules. Ideally such a system would be suitable for retrofitting into a manufacturing plant and would not require the use of expensive equipment, thus reducing capital expenditure. Ideally, such a system would be scalable to meet the demands of commercial manufacturing aspirations.
It is also an aim of the present invention to provide a system for processing, purifying and/or storing biomolecules faster and more simply than the prior art. Ideally the system would reduce waste produced from processing, purifying and/or storing biomolecules and therefore reduce the environmental impact. Ideally such a system would produce robust and reproducible results. Ideally such a system would be easy to operate without specialist knowledge. Ideally the system would be applicable to both batch and flow processing techniques.
It is also an aim of the present invention to provide a system for storing biomolecules in a safe, concentrated and contained manner for short or prolonged periods. Additionally, an aim of the present invention is to provide a system that allows the release of the immobilised biomolecule on demand.
This invention provides a system that achieves one or more of the above aims.